There is a growing literature on the role of dietary and environmental modulators in the control of reactive oxygen species that are implicated in the etiology of multiple and complex diseases such as cardiovascular diseases, Alzheimer's disease, Parkinson's disease, arthritis, certain cancers as well as in aging and apoptosis. Glutathione is a key endogenous antioxidant that plays a major role in cellular defense against reactive oxygen species and xenobiotics. The biosynthesis of glutathione is comprised of: (1) the upstream trans-sulfuration pathway that provides an avenue for conversion of the essential amino acid, methionine, to cysteine, the limiting amino acid in glutathione synthesis; and (2) the downstream pathway representing the final 2 steps catalyzed by gamma-glutamyl-cysteine synthetase and glutathione synthetase. The upstream pathway is present only in tissues with the highest concentration of glutathione, whereas the downstream pathway is ubiquitous. Tissues lacking the upstream pathway depend on an exogenous supply of cysteine, and inter-organ transport of glutathione plays a key role in providing this nonessential amino acid to these cells. A number of vitamins (B6, B12 and folic acid) and cofactors (heme and Zn2+) converge at this metabolic focal point and could potentially affect glutathione synthesis and, therefore, cellular redox homeostasis. The research design of this project is guided by 3 specific aims: (1) to address how regulation of the up- and downstream pathways is coordinated by using a mouse knockout model for cystathionine beta-synthase, the first enzyme in the trans-sulfuration pathway. These studies will be a prelude to characterizing the effects of macro- (methionine, cysteine) and micronutrients (B6, B12 and folate) on modulating glutathione pools in the 3 genetic backgrounds (+/+, +/-and -/- cbs); (2) to elucidate the relative efficacies of the different genotypes in handling oxidative and xenobiotic challenges; and (3) to initiate studies to elucidate the molecular mechanism of redox regulation of gamma-glutamyl- cysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, by focusing on the properties of the regulatory subunit that the principal investigator has discovered is a member of the aldo-keto reductase superfamily.